共查询到20条相似文献,搜索用时 46 毫秒
1.
Debebe Gebremedhin Pierre Bonnet Andrew S. Greene Sarah K. England Nancy J. Rusch Julian H. Lombard David R. Harder 《Pflügers Archiv : European journal of physiology》1994,428(5-6):621-630
The cellular mechanisms mediating hypoxia-induced dilation of cerebral arteries have remained unknown, but may involve modulation of membrane ionic channels. The present study was designed to determine the effect of reduced partial pressure of O2, PO
2, on the predominant K+ channel type recorded in cat cerebral arterial muscle cells, and on the diameter of pressurized cat cerebral arteries. A K+-selective single-channel current with a unitary slope conductance of 215 pS was recorded from excised inside-out patches of cat cerebral arterial muscle cells using symmetrical KCl (145 mM) solution. The open state probability (NP
o) of this channel displayed a strong voltage dependence, was not affected by varying intracellular ATP concentration [(ATP]i) between 0 and 100 M, but was significantly increased upon elevation of intracellular free Ca2+ concentration ([Ca2+]i). Low concentrations of external tetraethylammonium (0.1–3 mM) produced a concentration-dependent reduction of the unitary current amplitude of this channel. In cell-attached patches, where the resting membrane potential was set to zero with a high KCl solution, reduction of O2 from 21% to < 2% reversibly increased the NP
o, mean open time, and event frequency of the Ca2+-sensitive, high-conductance single-channel K+ current recorded at a patch potential of + 20 mV. A similar reduction in PO2 also produced a transient increase in the activity of the 215-pS K+ channel measured in excised inside-out patches bathed in symmetrical 145 mM KCl, an effect which was diminished, or not seen, during a second application of hypoxic superfusion. Hypoxia had no effect on [Ca2+]i or intracellular pH (pHi) of cat cerebral arterial muscle cells, as measured using Ca2+- or pH-sensitive fluorescent probes. Reduced PO2 caused a significant dilation of pressurized cerebral arterial segments, which was attenuated by pre-treatment with 1 mM tetraethylammonium. These results suggest that reduced PO2 increases the activity of a high-conductance, Ca2+-sensitive K+ channel in cat cerebral arterial muscle cells, and that these effects are mediated by cytosolic events independent of changes in [Ca2+]i and pHi. 相似文献
2.
Virginia Soares Lemos Kenneth Takeda 《Pflügers Archiv : European journal of physiology》1995,430(4):534-540
We have proposed recently that a pertussistoxin-insensitive Ca2+ influx stimulated by Y2-type receptor activation in CHP-234 human neuroblastoma cells underlies increases in intracellular free Ca2+ concentration ([Ca2+]i) induced by neuropeptide Y (NPY), which were strictly dependent on extracellular Ca2+ and independent of internal Ca2+ stores. We describe here the actions of NPY in these same cells, using the activity of Ca2+-activated K+ channels as an indicator of [Ca2+]i. The elementary slope conductance of these channels was 110±3 pS (with an asymmetrical K+gradient), their activity was greatly increased by application of ionomycin, and they were reversibly blocked by 1 mM tetraethylammonium (TEA) and 100 nM charybdotoxin. Application of 100 nM NPY, in the presence but not in the absence of extracellular Ca2+, increased the channel open probability. ATP applied in the absence of external Ca2+ caused rises both in channel open probability and [Ca2+]i. Inositol trisphosphate production was stimulated by ATP but not by NPY. In outside-out patches, NPY increased channel open probability, indicating that NPY-associated Ca2+ influx does not require all the intracellular machinery present in intact cells. Channel activation by NPY was unaffected by the replacement of guanosine 5-triphosphate (GTP) by (guanosine 5-O-(2-thiodiphosphate) (GDP[S]), a non-hydrolysable GDP analogue, in the pipette internal solution, consistent with the lack of involvement of G-proteins in the coupling of Y2-type receptors to Ca2+ influx in CHP-234 cells. 相似文献
3.
Amy Tse Frederick W. Tse Bertil Hille 《Pflügers Archiv : European journal of physiology》1995,430(5):645-652
In rat pituitary gonadotropes, gonadotropin-releasing hormone (GnRH) stimulates rhythmic release of Ca2+ from stores sensitive to inositol 1,4,5-trisphosphate [Ins(1,4,5)P
3
], which in turn induces an oscillatory activation of apamin-sensitive Ca2+-activated K+ current, I
K(Ca). Since GnRH also activates protein kinase C (PKC), we investigate the action of PKC while simultaneously measuring intracellular Ca2+ concentration ([Ca2+]i) and I
K(Ca). Stimulation of PKC by application of phorbol 12-myristate 13-acetate (PMA) did not affect basal [Ca2+]i. However, PMA or phorbol 12,13-dibutyrate (PdBu), but not the inactive 4-phorbol 12,13-didecanoate (4-PDD), reduced the frequency of GnRH-induced [Ca2+]i oscillation and augmented the I
K(Ca) induced by any given level of [Ca2+]i. The slowing of oscillations and the enhancement of I
K(Ca) were mimicked by synthetic diacylglycerol (1,2-dioctanoyl-sn-glycerol) and could be induced during ongoing oscillations that had been initiated irreversibly in cells loaded with guanosine 5-O-(3-thio-triphosphate) (GTP-[S]). In contrast, when oscillations were initiated by loading cells with Ins(1,4,5)P
3, phorbol esters enhanced I
K(Ca) without affecting the frequency of oscillation. The protein kinase inhibitor, staurosporine, reduced I
K(Ca) without affecting [Ca2+]i and partially reversed the phorbol-ester-induced slowing of oscillation. Therefore, activation of PKC has two rapid effects on gonadotropes. It slows [Ca2+]i oscillations probably by actions on phospholipase C, and it enhances I
K(Ca) probably by a direct action on the channels. 相似文献
4.
Carlos Villalobos Javier García-Sancho 《Pflügers Archiv : European journal of physiology》1996,431(3):371-378
Caffeine, a well known facilitator of Ca2+-induced Ca2+ release, induced oscillations of cytosolic free Ca2+ ([Ca2+]i) in GH3 pituitary cells. These oscillations were dependent on the presence of extracellular Ca2+ and blocked by dihydropyridines, suggesting that they are due to Ca2+ entry through L-type Ca2+ channels, rather than to Ca2+ release from the intracellular Ca2+ stores. Emptying the stores by treatment with ionomycin or thapsigargin did not prevent the caffeine-induced [Ca2+]i oscillations. Treatment with caffeine occluded phase 2 ([Ca2+]i oscillations) of the action of thyrotropin-releasing hormone (TRH) without modifying phase 1 (Ca2+ release from the intracellular stores). Caffeine also inhibited the [Ca2+]i increase induced by depolarization with high-K+ solutions (56% at 20 mM), suggesting direct inhibition of the Ca2+ entry through voltage-gated Ca2+ channels. We propose that the [Ca2+]i increase induced by caffeine in GH3 cells takes place by a mechanism similar to that of TRH, i.e. membrane depolarization that increases the firing frequency of action potentials. The increase of the electrical activity overcomes the direct inhibitory effect on voltage-gated Ca2+ channels with the result of increased Ca2+ entry and a rise in [Ca2+]i. Consideration of this action cautions interpretation of previous experiments in which caffeine was assumed to increase [Ca2+]i only by facilitating the release of Ca2+ from intracellular Ca2+ stores. 相似文献
5.
U. M. Rose R. J. M. Bindels A. Vis J. W. C. M. Jansen C. H. Van Os 《Pflügers Archiv : European journal of physiology》1993,423(5-6):378-386
Ca2+ channel blockers (CCB) have been shown to be protective against ischaemic damage of the kidney, suggesting an important role for intracellular Ca2+ ([Ca2+]i) in generating cell damage. To delineate the mechanism behind this protective effect, we studied [Ca2+]i in cultured proximal tubule (PT) cells during anoxia in the absence of glycolysis and the effect of methoxyverapamil (D600) and felodipine on [Ca2+]i during anoxia. A method was developed whereby [Ca2+]i in cultured PT cells could be measured continuously with a fura-2 imaging technique during anoxic periods up to 60 min. Complete absence of O2 was realised by inclusion of a mixture of oxygenases in an anoxic chamber. [Ca2+]i in PT cells started to rise after 10 min of anoxia and reached maximal levels at 30 min, which remained stable up to 60 min. The onset of this increase and the maximal levels reached varied markedly among individual cells. The mean values for normoxic and anoxic [Ca2+]i were 118±2 (n=98) and 662±22 (n=160) nM, respectively. D600 (1 M), but not felodipine (10 M), significantly reduced basal [Ca2+]i in normoxic incubations. During anoxia 1 M and 100 M D 600 significantly decreased anoxic [Ca2+]i levels by 22 and 63% respectively. Felodipine at 10 M was as effective as 1 M D600. Removal of extracellular Ca2+ and addition of 0.1 mM La3+ completely abolished anoxia-induced increases in [Ca2+]i. We conclude that anoxia induces increases in [Ca2+]i in rabbit PT cells in primary culture, which results from Ca2+ influx. Since this Ca2+ influx is partially inhibited by low doses of CCBs, Ltype Ca2+ channels may be involved. 相似文献
6.
T. Leinders R. G. D. M. van Kleef H. P. M. Vijverberg 《Pflügers Archiv : European journal of physiology》1992,422(3):217-222
Effects of Cd2+, Co2+, Fe2+ and Mg2+ (1 M and 100 M) and Pb2+ (1 M and 90 M) on single-channel properties of the small-conductance (SK) and large-conductance (BK) Ca2+-activated K+ channels were investigated in inside-out patches of N1E-115 mouse neuroblastoma cells. Cd2+, Co2+ and Pb2+, but not Fe2+ and Mg2+, cause SK channel opening. The potency of the metals in enhancing the SK channel-open probability follows the sequence Cd2+ Pb2+>Ca2+>Co2+ Mg2+, Fe2+. The four metals that cause SK channel opening are equipotent in enhancing the opening frequency of SK channels. The BK channel is activated by Pb2+ and Co2+, whereas Cd2+, Fe2+ and Mg2+ are ineffective. The potency of the metals in enhancing BK channel-open probability, open time and opening frequency follows the sequence Pb2+>Ca2+>Co2+Cd2+, Mg2+, Fe2+. The results show that SK channels are much more sensitive to Cd2+ than BK channels and indicate that Cd2+ is a selective agonist of SK channels. It is concluded that the various metal ions bind to the same regulatory site(s) at which Ca2+ activates the SK and BK channels under physiological conditions. The different potency sequences of metal ions with respect to BK and SK channel activation indicate that the regulatory sites of these Ca2+-activated K+ channeles have distinct chemical and physical properties. 相似文献
7.
Microfluorimetric studies were carried out to investigate the effects of hypoosmotic swelling on intracellular Ca2+ concentration ([Ca2+]i) in single rat epididymal cells. In Ca2+-free solution containing 50 mol/l ethylenebis(oxonitrilo)tetraacetate (EGTA) hypoosmotic swelling (–160 mosmol/l) induced a transient rise in [Ca2+]i which was either monophasic, biphasic or oscillatory. The [Ca2+]i responses to repeated hypoosmotic stimulations followed a decremental pattern. However, if 2.5 mmol/l Ca2+ was admitted during the recovery period between successive stimulations, the second and the third [Ca2+]i responses were slightly greater than the first. Increasing the change in osmolarity from –14±1.0 to –154±1.5 mosmol/l increased the rise in [Ca2+]i but reduced the [Ca2+]i response to subsequent ionomycin stimulation (4 mol/l). The swelling- and the ionomycin-induced rises in [Ca2+]i followed a reciprocal pattern. It was suggested that intracellular Ca2+ release in response to cell swelling in the epididymal epithelium might play a role in cell volume regulation and the control of epididymal fluid osmolarity. 相似文献
8.
W. V. Breuer E. Mack A. Rothstein 《Pflügers Archiv : European journal of physiology》1988,411(4):450-455
In dissociated MDCK cells, activators of the cyclic AMP system cause depolarization detectable by changes in fluorescence of the membrane potential sensitive dye bisoxonol. Addition of forskolin (60 M), vasopressin (2 M), 8-bromo-cyclic AMP (0.5 mM) or l-epinephrine (10 M) depolarized the cells substantially in low Cl– (5 mM) but had little effect in high Cl– (140 mM) solution. These results are consistent with cyclic AMP activation of Cl– channels. The Ca2+-ionophore ionomycin (1 M) produced a rapid hyperpolarization in low and high Cl– solutions, consistent with K+ channel opening. Using a clonal subline, MDCK-14, the magnitude of the ionomycin hyperpolarization was roughly proportional to the concomitant rise in [Ca2+]i as measured with the intracellular Ca2+ probe indo-I. Both l-epinephrine and isoproterenol appeared to activate the Cl– channels. However only l-epinephrine produced a [Ca2+]i rise and a transient hyperpolarization (due to K+ channel opening), which preceeded the depolarization due to Cl– channel opening. The l-epinephrine-induced [Ca2+]i response of the heterogeneous MDCK cell population but not of the clonal subline MDCK-14 was inhibited by removal of extracellular Ca2+. In the latter only the slow secondary phase of the [Ca2+]i rise was affected by Ca2+ removal. It is concluded that l-epinephrine activates K+ and Cl– channels in a sequential manner in MDCK cells by Ca2+ and cAMP signals, presumably via - and -adrenergic receptors located on the same cell.Abbreviations MDCK cells
Madin Darby Canine Kidney cells
- [Ca2+]i
intracellular calcium concentration
- [Cl–]i
intracellular chloride concentration
- [Cl–]o
extracellular chloride concentration
- [Na++K+]i
intracellular concentration of Na+ and K+
- [Na++K+]o
extracellular concentration of Na+ and K+
- EM
transmembrane potential
- ECl
chloride equilibrium potential
- EK
potassium equilibrium potential
- bis-oxonol
[bis(1,3-diethylthio-barbiturate)] trimethine oxonol
- DMSO
dimethylsulfoxide
- EDTA
ethylenediaminetetraacetic acid
- EGTA
ethylene glycol bis (-aminoethyl ether) N,N-tetraacetic acid
- Hepes
4-(2-hydroxyethyl)-1 piperazineethanesulfonic acid
- NMG+
N-methylglucamine
- RPMI medium
Rosewell Park Memorial Institute medium 相似文献
9.
Ca2+-activated maxi K+ channels were studied in inside-out patches from smooth muscle cells isolated from either porcine coronary arteries or guinea-pig urinary bladder. As described by Groschner et al. (Pflügers Arch 417:517, 1990), channel activity (NP
o) was stimulated by 3 M [Ca2+]c (1 mM Ca-EGTA adjusted to a calculated pCa of 5.5) and was suppressed by the addition of 1 mM Na2ATP. The following results suggest that suppression of NP
o by Na2ATP is due to Ca2+ chelation and hence reduction of [Ca2+]c and reduced Ca2+ activation of the channel. The effect was absent when Mg ATP was used instead of Na2ATP. The effect was diminished by increasing the [EGTA] from 1 to 10 mM. The effect was absent when [Ca2+]c was buffered with 10 mM HDTA (apparent pK
Ca 5.58) instead of EGTA (pK
Ca 6.8). A Ca2+-sensitive electrode system indicated that 1 mM Na2ATP reduced [Ca2+]c in 1 mM Ca-EGTA from 3 M to 1.4 M. Na2ATP, Na2GTP, Li4AMP-PNP and NaADP reduced measured [Ca2+]c in parallel with their suppression of NP
o. After the Na2ATP-induced reduction of [Ca2+]c was re-adjusted by adding either CaCl2 or MgCl2, the effect of Na2ATP on NP
o disappeared. In vivo, intracellular [Mg2+] exceeds free [ATP4–], hence ATP modulation of maxi K+ channels due to Ca2+ chelation is without biological relevance. 相似文献
10.
H. Lerche Ch. Fahlke P. A. Laizzo F. Lehmann-Horn 《Pflügers Archiv : European journal of physiology》1995,429(5):738-747
Ca2+-activated K+ channels of a large conductance (BKCa) in human skeletal muscle were studied by patch clamping membrane blebs and by using the three microelectrode voltage-clamp recording technique on resealed fibre segments. Single-channel recordings in bleb-attached and inside-out modes revealed BKCa conductances of 230 pS for symmetrical and 130 pS for physiological K+ distributions. Open probability increased with membrane depolarization and increasing internal [Ca2+]. The Hill coefficient was 2.0, indicating that at least two Ca2+ ions are required for full activation. Kinetic analysis revealed at least two open and three closed states. An additional long-lived inactivated state, lasting about 0.5–20 s, was observed following large depolarizations, when extracellular K+ was lowered to physiological values. BKCa were blocked by three means: (1) externally by tetraethylammonium which reduced single-channel amplitude (IC50 approx. 0.3 mM); (2) internally by polymyxin B which decreased the open probability (IC50 approx. 5 g/ml); and (3) externally by charybdotoxin which caused long-lasting periods of inactivation (IC50 <10 nM). Measurements on resealed fibre segments at physiological [K+] were in accordance with the single-channel data: only when intracellular [Ca2+] was elevated did charybdotoxin (50 nM) reduce the macroscopic membrane K+ conductance with depolarizing voltage steps. 相似文献
11.
Katsuhisa Ikeda Yoshitaka Saito Akinori Nishiyama Tomonori Takasaka 《Pflügers Archiv : European journal of physiology》1992,420(5-6):493-499
The outer hair cell isolated from the guinea-pig was superfused in vitro and the cytosolic calcium concentration ([Ca2+]i) and sodium concentration ([Na+]i) were measured using fluorescence indicators. Under the resting condition, [Ca2+]i and [Na+]i were 91±9 nM (n = 51) and 110±5 mM (n = 12), respectively. Removal of external Na+ by replacing with N-methyl-D-glucamine (NMDG+) increased [Ca2+]i by 270±79% (n = 27) and decreased [Na+]i by 23±4 mM (n = 6). Both changes in [Ca2+]i and [Na+]i were totally reversible on returning external Na+ to the initial value and were inhibited by addition of 0.1 mM La3+ or 100 M amiloride 5-(N,N-dimethyl) hydrochloride. Elevation of external Ca2+ ions to 20 mM reversibly decreased [Na+]i by 8±6 mM (n = 5). Moreover, the chelation of the intracellular Ca2+ with 1,2-bis (2-aminophenoxy) ethane-N,N,N,N-tetraacetic acid (BAPTA) exerted an inhibitory action on the NMDG+-induced reduction in [Na+]i. Exposure to 5 mM NaCN for 2 min significantly and reversibly increased [Ca2+]i by 290±37% (n = 5), but did not affect the [Ca2+]i elevation induced by the NMDG+ solution. The rise in [Ca2+]i induced by the NMDG+ solution was not enhanced by ouabain pretreatment. Addition of ouabain did not alter the [Na+]i. The present results are best explained by the presence of an Na+-Ca2+ exchanger in cell membrane and indicate that the activity of Na+/K+ pump is poor in outer hair cells. 相似文献
12.
Agonist-dependent modulation of Ca2+ sensitivity in rabbit pulmonary artery smooth muscle 总被引:10,自引:0,他引:10
B. Himpens T. Kitazawa A. P. Somlyo 《Pflügers Archiv : European journal of physiology》1990,417(1):21-28
The effects of the stable thromboxane analogue U46619, the
1-adrenergic agent phenylephrine and depolarization with high K+ on cytoplasmic Ca2+ ([Ca2+]i) and force development were determined in rabbit pulmonary artery smooth muscle. Following stimulation with each of the excitatory agents, the time course of the [Ca2+]i/force relationship described counter-clockwise hysteresis loops with the rise and fall in [Ca2+]i leading, respectively, contraction and relaxation. The rank order of the force/[Ca2+]i ratios evoked by the different methods of stimulation was: U46619 > phenylephrine high K+. The difference between the actions of U46619 and phenylephrine was due to the lesser Ca2+-releasing and greater Ca2+-sensitizing action of U46619. Both U46619 and phenylephrine also released intracellular Ca2+ in intact (non-permeabilized) preparations. The effects of the two agonists on force, at constant free cytoplasmic [Ca2+] maintained with EGTA, were also determined in preparations permeabilized with staphylococcal -toxin, in which intracellularly stored Ca2+ was eliminated with A23187. Sensitization of the contractile response to Ca2+ by agonists was indicated by the contractile responses of permeabilized muscles to U46619 and to phenylephrine, in the presence of constant, highly buffered [Ca2+]i. These contractions were inhibited by GDP[S] and could also be elicited by GTP. We conclude that, in addition to changing [Ca2+]i, pharmacomechanical coupling can also modulate contraction by altering the sensitivity of the regulatory/contractile apparatus of smooth muscle to [Ca2+]i, through a G-protein-coupled mechanism. 相似文献
13.
Single channel Ca2+ currents inHelix pomatia neurons 总被引:4,自引:0,他引:4
Unitary Ca2+ currents of TEA injected Helix neurons were recorded in the Giga seal situation (6, 7) from microscopic membrane patches exposed to 50 mM [Ca2+]o, O [Na+]o, 20 mM [TEA+]o and 2.5 M [TTX]o. Constant field assumptions yield a channel permeability of 2.9±1.0×10–14 cm3s–1 corresponding to slope conductances of 5 to 15 pS between 0 and –30 mV. Frequency of occurrence of the units strongly increased with depolarization. Mean open time of the Ca2+ channels was about 3 ms without obvious dependence on voltage. A similar open time was seen with [Ba2+]o, yielding about double the current strength when compared with [Ca2+]o. 相似文献
14.
A newly identified Ca2+ dependent K+ channel in the smooth muscle membrane of single cells dispersed from the rabbit portal vein 总被引:11,自引:0,他引:11
R. Inoue K. Okabe K. Kitamura H. Kuriyama 《Pflügers Archiv : European journal of physiology》1986,406(2):138-143
We found a new type of Ca2+-dependent K+ channel in smooth muscle cell membranes of single cells of the rabbit portal vein. A slope conductance of the current was 180 pS when 142 mM K+ solution was exposed to both sides of the membrane (this channel was named the KM channel, in comparison to the known KL and KS channels from the same membrane patch; Inoue et al. 1985). This KM channel was less sensitive to the cytoplasmic Ca2+ concentration, [Ca2+]i, but was sensitive to the extracellular Ca2+, [Ca2+]o, e.g. in the outside-out membrane patch, lowering the [Ca2+]o in the bath markedly reduced the open probability of this channel, and also in cell-attached configuration, lowering of the [Ca2+]o using the internally perfused patch clamp electrode device reduced the opening of KM channel. TEA+ (1–10 mM) reduced the amplitude of the elementary current through the KM channel applied from each side of the membrane, but this agent inhibited the KM channel to a greater extent when applied to the inner than to the outer surface of the membrane. Furthermore, this KM channel had a weak voltage dependency, and the open probability of the channel remained much the same within a wide range of potential (from –60 mV to +60 mV). Whereas most Ca2+-dependent K+ channels are regulated mainly by [Ca2+]i and possess a voltage dependency, these properties of the KM channel differed from other Ca2+-dependent K+ channels. The elucidation of this KM channel should facilitate explanations of the actions of external Ca2+ or TEA+ on the membrane potential, in the smooth muscles of the rabbit portal vein. 相似文献
15.
Gervaise Loirand Pierre Pacaud 《Pflügers Archiv : European journal of physiology》1995,430(3):429-436
Effects of exogenous adenosine 5-triphosphate (ATP) were studied by measurements of intracellular Ca2+ concentration ([Ca2+]i) and membrane currents in myocytes freshly isolated from the human saphenous vein. At a holding potential of –60 mV, ATP (10 M) elicited a transient inward current and increased [Ca2+]i. These effects of ATP were inhibited by ,-methylene adenosine 5-triphosphate (AMPCPP, 10 M). The ATP-gated current corresponded to a non-selective cation conductance allowing Ca2+ entry. The ATP-induced [Ca2+]i rise was abolished in Ca2+-free solution and was reduced to 30.1±5.5% (n=14) of the control response when ATP was applied immediately after caffeine, and to 23.7±3.8% (n=11) in the presence of thapsigargin. The Ca2+-induced Ca2+ release blocker tetracaine inhibited the rise in [Ca2+]i induced by both caffeine and ATP, with apparent inhibitory constants of 70 M and 100 M, respectively. Of the ATP-induced increase in [Ca2+]i 29.3±3.9% (n=8) was tetracaine resistant. It is concluded that the effects of ATP in human saphenous vein myocytes are only mediated by activation of P2x receptor channels. The ATP-induced [Ca2+]i rise is due to both Ca2+ entry and Ca2+ release activated by Ca2+ ions that enter the cell through P2x receptor channels. 相似文献
16.
Peptide hormones control salt reabsorption in cortical thick ascending limb (cTAL) cells of the loop of Henle. These agonists act, in part, through alterations on intracellular Ca2+ ([Ca2+]i). Primary cell cultures were prepared from porcine kidneys using a double antibody technique (goat antihuman Tamm-Horsfall and rabbit antigoat IgG antibodies). [Ca2+]i was determined in single cells with fluorescent techniques using fura-2. Parathyroid hormone (PTH) and arginine vasopressin (AVP) transiently increased [Ca2+]i in a dose-dependent manner. [Ca2+]i maximally increased from 85±5 nmol/l to 608±99 nmol/l with PTH, 10–6M, and to 766±162 nmol/l with AVP, 10–7 M. The increment in [Ca2+]i by both hormones was by intracellular Ca2+ release and entry through plasma membrane Ca2+ channels. 8-Bromoadenosine-3, 5-cyclic monophosphate (8-BrcAMP), 10–4M, increased [Ca2+]i(basal 83±3 to 427±121 nmol/l) but only from internal sources as nifedipine (10 mol), ([Ca2+]i changes: 86±4 to 390±29 nmol/l) and removal of bath Ca
o
2+
, ([Ca2+]ichanges: 84±6 to 517±142 nmol/l), were without effect on agonist-induced [Ca2+]i. Thapsigargin, 1.5 mol, completely abolished the AVP- and cyclic adenosine monophosphate-(cAMP)-induced Ca2+ transients, and partially inhibited PTH-mediated Ca2+ transients by about 50%. Pretreatment with 8-BrcAMP inhibited the PTH and AVP responses likely through depletion of cAMP-sensitive Ca2+ stores. Activation of protein kinase C (PKC) with phorbol esters inhibited PTH and AVP responses and 8-BrcAMP-induced [Ca2+]i transients. The responses partially returned following down-regulation of PKC with prolonged exposure to phorbol esters. These data suggest that PKC activation modulates agonist-induced Ca2+ release and entry, possibly through actions on intracellular release mechanisms. In summary, PTH and AVP stimulate Ca2+ signals by similar pathways involving cAMP and inositol 1,3,4-trisphosphate activity at similar sites on the endoplasmic reticulum and plasma membrane. These results suggest that peptide hormones may act through Ca2+ and be modulated by different pathways which may have diverse effects on cTAL function. 相似文献
17.
Antonio R. Artalejo Antonio G. García Erwin Neher 《Pflügers Archiv : European journal of physiology》1993,423(1-2):97-103
Simultaneous whole-cell patch-clamp and fura-2 fluorescence [Ca2+]i measurements were used to characterize Ca2+-activated K+ currents in cultured bovine chromaffin cells. Extracellular application of histamine (10 M) induced a rise of [Ca2+]i concomitantly with an outward current at holding potentials positive to –80 mV. The activation of the current reflected an increase in conductance, which did not depend on membrane potential in the range –80 mV to –40 mV. Increasing the extracellular K+ concentration to 20 mM at the holding potential of –78 mV was associated with inwardly directed currents during the [Ca2+]i elevations induced either by histamine (10 M) or short voltage-clamp depolarizations. The current reversal potential was close to the K+ equilibrium potential, being a function of external K+ concentration. Current fluctuation analysis suggested a unit conductance of 3–5 pS for the channel that underlies this K+ current. The current could be blocked by apamin (1 M). Whole-cell current-clamp recordings snowed that histamine (10 M) application caused a transient hyperpolarization, which evolved in parallel with the [Ca2+]i changes. It is proposed that a small-conductance Ca2+-activated K+ channel is present in the membrane of bovine chromaffin cells and may be involved in regulating catecholamine secretion by the adrenal glands of various species. 相似文献
18.
Paul E. Squires Brigitte Amiranoff Mark J. Dunne 《Pflügers Archiv : European journal of physiology》1994,428(3-4):275-282
Single-cell microfluorimetry techniques have been used to examine the effects of acetylcholine (0.1–100 M) on the intracellular free calcium ion concentration ([Ca2+]i) in a human-derived pancreatic somatostatin-secreting cell line, QGP-1N. When applied to the bath solution, acetylcholine was found to evoke a marked and rapid increase in [Ca2+]i at all concentrations tested. These responses were either sustained, or associated with the generation of complex patterns of [Ca2+]i transients. Overall, the pattern of response was concentration related. In general, 0.1–10 M acetylcholine initiated a series of repetitive oscillations in cytoplasmic Ca2+, whilst at higher concentrations the responses consisted of a rapid rise in [Ca2+]i followed by a smaller more sustained increase. Without external Ca2+, 100 M acetylcholine caused only a transient rise in [Ca2+]i, whereas lower concentrations of the agonist were able to initiate, but not maintain, [Ca2+]i oscillations. Acetylcholine-evoked Ca2+ signals were abolished by atropine (1–10 M), verapamil (100 M) and caffeine (20 mM). Nifedipine failed to have any significant effect upon agonist-evoked increases in [Ca2+]i, whilst 50 mM KCl, used to depolarise the cell membrane, only elicited a transient increase in [Ca2+]i. Ryanodine (50–500 nM) and caffeine (1–20 mM) did not increase basal Ca2+ levels, but the Ca2+-ATPase inhibitors 2,5-di(tert-butyl)-hydroquinone (TBQ) and thapsigargin both elevated [Ca2+]i levels. These data demonstrate for the first time cytosolic Ca2+ signals in single isolated somatostatin-secreting cells of the pancreas. We have demonstrated that acetylcholine will evoke both Ca2+ influx and Ca2+ mobilisation, and we have partially addressed the subcellular mechanism responsible for these events. 相似文献
19.
Hideaki Katsuyama Shinichi Ito Take Itoh Hirosi Kuriyama 《Pflügers Archiv : European journal of physiology》1991,419(5):460-466
To study the essential features of acetylcholine (ACh)-and caffeine-sensitive cellular Ca2+ storage sites in single vascular smooth muscle cells of the porcine coronary artery, the effects of ryanodine on both ACh- and caffeine-induced Ca2+ mobilization were investigated by measuring intracellular Ca2+ concentration ([Ca2+]i) using Fura 2 in Ca2+-containing or Ca2+-free solution. The resting [Ca2+]i of the cells was 122 nM in normal physiological solution and no spontaneous activity was observed. In a solution containing 2.6 mM Ca2+, 10 M ACh or 128 mM K+ produced a phasic, followed by a tonic, increase in [Ca2+]i but 20 mM caffeine produced only a phasic increase. In Ca2+-free solution containing 0.5 mM ethylenebis(oxonitrilo)tetraacetate (EGTA), the resting [Ca2+]i rapidly decreased to 102 nM within 5 min, and 10 M ACh or 20 mM caffeine (but not 128 mM K+) transiently increased [Ca2+]i. Ryanodine (50 M) greatly inhibited the phasic increase in [Ca2+]i induced by 10 M ACh or 5 mM caffeine and increased the time to peak and to the half decay after the peak in the presence or absence of extracellular Ca2+. By contrast, ryanodine (50 M) enhanced the tonic increase in [Ca2+]i induced by 128 mM K+ and also by 10 M ACh in Ca2+-containing solution. In Ca2+-free solution containing 0.5 mM EGTA, ACh (10 M) failed to increase [Ca2+]i following application of 20 mM caffeine. The level of [Ca2+]i induced by 20 mM caffeine was greatly reduced, but not abolished, following application of 10 M ACh in Ca2+-free solution. These results suggest that both ACh and caffeine release Ca2+ from the ryanodine-sensitive sarcoplasmic reticulum (SR) in smooth muscle cells of the porcine coronary artery. The finding that ryanodine significantly increased the resting [Ca2+]i and inhibited the rate of decline of [Ca2+]i following wasthout of high K+ or ACh in Ca2+-containing solution suggests that SR may negatively regulate the resting [Ca2+]i in smooth muscle cells of the porcine coronary artery. 相似文献
20.
Emil C. Toescu Ole H. Petersen 《Pflügers Archiv : European journal of physiology》1994,427(3-4):325-331
In pancreatic acinar cells, as in many other cell types, the tumour promoter thapsigargin (TG) evokes a significant increase
of intracellular free Ca2+ ([Ca2+]i). The increases of [Ca2+]i evoked by TG was associated with significant changes of plasma membrane Ca2+ permeability, with [Ca2+]i values following changes in extracellular [Ca2+]. Plasma membrane Ca2+ extrusion is activated rapidly as a consequence of the rise in [Ca2+]i evoked by TG and the rate of extrusion is linearly dependent on [Ca2+]i up to 1 μM Ca2+. In contrast, the activation of the Ca2+ entry pathway is delayed and the apparent rate of Ca2+ entry is independent of [Ca2+]i. In the presence of 20 mM caffeine, which reduces the resting levels of inositol trisphosphate (InsP3), the increase of [Ca2+]i evoked by TG was significantly reduced. The reduction was manifest both as a decrease of the amplitude of the [Ca2+]i peak (30% reduction) and, more importantly, as a reduction of the apparent maximal rate of [Ca2+]i increase (from 12.3±1.0 to 6.1±0.6 nM Ca2+/s). The inhibition evoked by caffeine was reversible and the removal of caffeine in the continuous presence of TG evoked
a further increase of [Ca2+]i. The amplitude of the [Ca2+]i increase upon caffeine removal was reduced as a function of the time of TG exposure. Addition of TG in the presence of 1
mM La3+, which is known to inhibit the plasma membrane Ca2+-activated adenosine triphosphatase, induced a much higher peak of [Ca2+]i. This increase was associated with an augmentation of the apparent rate of [Ca2+]i increase (from 12.3±1.2 to 16.1±1.9 nM Ca2+/s). The inhibitory effect of caffeine, as well as the increase in [Ca2+]i observed on caffeine removal was not affected by the presence of 1 mM La3+. These data indicate that an important component of the TG-evoked [Ca2+]i increase is due to InsP3-sensitive Ca2+ release which is probably mediated by the resting levels of InsP3. 相似文献